UPAR mimicking peptide

ABSTRACT

Peptides comprising the SRSRY sequence (SEQ ID NO: 7) derived from the uPAR (urokinase receptor) are endowed with chemotactic activity, making them useful for preventing or activating the migration of cells in a mammal. More particularly, the peptides of the invention are useful for the treatment of cancer, autoimmune diseases, and/or hyperinflammatory diseases and for stimulating wound healing.

This application is the U.S. national phase of PCT EP98/01547 filed Mar.18, 1998, which claims priority to U.S. Provisional application No.60/041,112, filed Mar. 20, 1997.

FIELD OF THE INVENTION

The present invention relates to a method for preventing or activatingthe migration of cells in a mammal, in particular a human. The methodcomprises the use of synthetic peptides, or of defined recombinantsoluble forms of the urokinase receptor to activate the recruitment ofinflammatory cells in a mammal, by stimulating a cellular adaptor thatmediates the chemotactic activity of uPA. This mechanism does notrequire the protease activity of urokinase. Actually it by-passes it byacting on a downstream step, namely the interaction of such a peptide orrecombinant soluble form of urokinase receptor with a cellular adaptor.Additionally, the reagents produced are also useful to identify andisolate drugs that can inhibit said processes of cell recruitment, andhence may be employed in blocking the malignant phenotype of cancer andof aggressive hyper-or auto-inflammatory diseases.

GENERAL BACKGROUND

Cell Migration and Disease

The response of the human body to noxic stimuli is dependent on thelocal production of inflammatory molecules as well as on the recruitmentof specialized cells that migrate from the blood and the neighbouringtissue into the damaged site. These cells, neutrophils,monocyte-macrophages, lymphocytes and endothelial cells, respond tospecific migratory stimuli and build up a defense response that resultsin the destruction and elimination of the nocive agent or organism.While essential and beneficial for the host defense, this type ofresponse may also prove dangerous for the host or may hamper the successof some therapeutic or preventive approaches like transplantation andvaccination. In fact, a series of severe pathological entities existwhich can be ascribed to excess or deficient migratory cell response.For example, immunodeficient patients fail to respond to infectiveagents and this may be due to inability of the cells to rush to thedamaged or infected site. Excess of recruitment, on the other hand, maybe responsive of destructive pathologies in which defense cells attackcells and functions of the host, as it happens for example in autoimmunediseases. Failure to properly respond to vaccination, moreover, may alsodepend on unwanted recruitment of host inflammatory cells that destroythe antigenic cells too quickly to promote an immunological response.The availability of a specific “adjuvant” to natural immunity cells,would be advantageous as it would produce a stronger antibody response.

The malignancy of cancer cells mostly consists in the ability to invadeand metastasize at a distance; in this process, however, non-malignantstromal host cells actively participate in establishing the malignantphenotype and hence the destructive and invasive phenotype. While themechanisms involved are largely unknown, it is clear that stromal cellsnot only respond to stimuli arriving from the cancer cells but alsosignal to cancer cells through mechanisms of their own. In order toblock the invasive phenotype of cancer, it is thus essential to act bothon cancer and on stromal cells.

The Urokinase/urokinase Receptor System

The activity of urokinase (uPA) can be confined to the cell surface bythe presence of a specific receptor (uPAR, CD87). uPA is a serineprotease important in maintaining the fibrinolytic state of the body asit generates plasmin from plasminogen and hence prevents fibrindeposition. Plasmin is a broad spectrum protease that can destroy manyproteins of the extracellular matrix and hence the inter-cellular andcell-to-extracellular matrix connections. UPA and uPAR have long beenrecognised as regulators of cell migration and hence to be important ininflammation and cancer invasion. In addition to functions connected toits proteolytic activity, uPA has other properties that do not requireproteolytic activity, but simply the binding to the receptor. In fact,the proteolytic and the receptor binding activities are separated on theuPA molecule and can be assayed individually (receptor binding in theamino terminal fragment, proteolysis in the carboxy-terminal fragment).Among the functions that do not require the proteolytic moiety of uPA,are the stimulation of mitogenesis, cell migration, adhesion and, inparticular, chemotaxis (Gudewicz and Bilboa, 1987; Gyetko et al., 1994;Resnati et al., 1996; Besser et al., 1996).

The specific uPA receptor (uPAR) is a GPI-anchored plasma membraneprotein endowed with a very high affinity (Kd of 0.1-1 nM) for uPA,pro-uPA and inhibited forms of uPA, like the uPA-PAI-1 complex (Fazioliand Blasi, 1994). In addition to uPA, uPAR also binds vitronectin withan about 10-20 nM affinity as well as integrins. Structurally, uPAR isformed by three repeats of about 90 amino acid residues connected by twolinker regions (Danø, Blasi et al., 1990; Behrendt et al., 1991) whichdefine functionally and structurally different domains: the aminoterminal domain (D1) contains the uPA binding site, while thecarboxyterminal region containing domains D2 and D3 binds vitronectin.However, the integrity of the three-domains structure is necessary forhigh affinity binding to uPA at least with the purified, soluble protein(Danø et al., 1994). To date, no information has been provided as to thefunction of the linker regions.

uPAR is expressed in circulating blood cells, in particular monocytes,neutrophils and T-lymphocytes but not in erythrocytes or B-lymphocytes.,In addition, uPAR is a target gene in lymphocytes and macrophageactivation (CD87). Indeed, monocytes and monocyte-like cells (like HL60,U937) express uPAR, or are induced to overexpress uPAR by a variety ofcytokines and other agents, like phorbol ester PMA, phytohemagglutinin,bacterial liposaccharide, TGFb1/vitamin D3, GM-CSF, IFNg, TNFa andothers. In human T-lymphocytes, stimulation of the TCR/CD3 complex,lymphokines IL2, IL4, IL7 or the concomitant activation of the T cellreceptor and integrins engagement, all induce uPAR expression (Nykjmr etal., 1994; Bianchi et al., 1996). It is also noteworthy, that tumorinfiltrating T-lymphocytes heavily express UPA-R and some of theproperties of the activated T-lymphocytes, in particular their migrationthrough reconstituted basement membranes, appear to be at least in partuPA- and uPAR-dependent (Bianchi et al., 1996). In view of the importantcontribution of stromal cells to the invasiveness of cancer, it is alsoimportant to stress that macrophages present in human breast cancer andother cancers, where the level of uPAR production by the overall tumorimportantly contributes to its malignancy, express high levels of uPAR(Brunner et al., 1996). The cooperation between stromal and cancer cellsin cancer invasiveness, implies that high expression of uPAR by canceror stromal cells, can affect cell prognosis possibly through differentmechanisms depending on the overexpressing cell-type.

uPA/uPAR and Chemotaxis

Cooperation between cancer and stromal cells poses the problem of howuPAR expression on stromal cells influences the malignancy of cancercells. Since the uPA/uPAR system has chemotactic activity, theproduction of uPAR by stromal cells can attract cancer cells through achemokine like action. Indeed, chemokines produced by certain cells areanchored to a presentation molecule in the extracellular matrix andattract other cells having specific receptors (Schall and Bacon, 1996).The information available on the uPA/uPAR system supports thispossibility.

DISCLOSURE OF THE INVENTION

uPA/uPAR and Chemotaxis

Stimulation of chemotaxis in monocyte-like cells, fibroblasts and somecancer cells requires the specific cell surface uPAR (CD87) whichmediates the chemoattractant activity of uPA (Resnati et al., 199.6).Chemotaxis by uPA does not require its protease activity, but only theoccupancy of its receptor, and can be reproduced with the enzymaticallyinactive receptor binding moiety ATF (amino terminal fragment), pro-uPAand by chymotrypsin-cleaved soluble uPAR (Resnati et al., 1996). Inmouse, the importance of this system in cell recruitment in vivo isshown by the fact that uPA is essential for the inflammatory response;indeed, mice lacking the uPA gene are highly defective in the responseto bacterial and possibly other infections, and are incapable ofrecruiting T-lymphocytes and macrophages to the site of infection(Gyetko et al., 1996). In man, uPA and uPAR are induced during T cellsactivation and directly contribute to the in vitro migration of human Tcells (Nykjær et al., 15 1994; Bianchi et al.,1996). Moreover, tumorinfiltrating T-lymphocytes heavily express uPAR (Bianchi et al., 1996).

uPA-dependent chemotaxis is mediated by the activation of src-familytyrosine kinases (i.e. Hck in monocytes and Src in fibroblasts). Themechanism involved requires that uPA modifies uPAR conformation suchthat it can bind to a still unidentified adaptor. Indeed, in cellslacking uPAR, a mixture of fragments generated by cleaving a soluble,recombinant uPAR with chymotrypsin, has a very potent chemoattractantactivity (IC50 of 10-20 pM) (Resnati et al., 1996).

In Boyden chamber-based assays, uPA or its derivatives induce migrationthrough a chemotactic gradient in bovine adrenal capillary endothelialcells, keratinocytic cell lines, monocytes, monocyte-like cells,fibroblasts cell lines, and in neutrophils both in vitro and in vivo(Besser et al., 1996; Resnati et al., 1996). The involvement of uPAR inchemotaxis is not only a direct one, but may also be indirect byinterfering with the chemotaxis induced by other chemokines. Exposure ofhuman neutrophils to a chemotactic gradient of FMLP or MCP-1 chemokinelocalizes uPAR to the leading edge of the migrating cell; anti-uPARantibodies ablate the chemotactic activity. The chemotactic effect ofFMLP or MCP-1 does not require the proteolytic activity of uPA, but uPARoccupancy is absolutely required for chemotaxis as uPAR antibodies orantisense RNA expression totally abolishes FMLP or MCP-1 activity.

The steps between the binding of chemokines to chemokine receptors andcell movements, are many and complex: signal transduction,reorganization of cytoskeleton, formation of focal adhesions, attachmentand detachment from the substrate with pseudopodal extension andretraction to effect directional migration (Premack and Schall, 1996).The chemotactic activity of uPAR has many of the properties ofchemokines. Interaction between uPAR and integrins has been observed inneutrophils: the leucocyte integrin CD11b/CD18, aMb2, also known as thecomplement receptor type-3 (CR3) has been shown to physically associatewith uPAR. This interaction is reversible and correlates with cellshape. In resting cells, uPAR and CR3 are co-localized, but followingspontaneous cell polarization and migration, the two receptorsdissociate, CR3 concentrates in the uropods and uPAR in the lamellipodiaof polarized cells. Since CR3 regulates cell adhesion, chemotaxis andcell migration into inflammatory sites, the interaction with uPAR may becentral to all these functions and hence to the cell-recruitment actionof uPA/uPAR. No information however is available as to whether thisintegrin (or others) is directly involved in mediating the uPARchemotactic signal, i.e. whether this integrin is the so farunidentified adaptor.

Activation of migration by uPAR occupancy is observed also in epithelialcell lines where uPAR associates with a protein kinase that canserine-phosphorylate two cytokeratins, CK18 and CK8. The enzymeresponsible is possibly the protein kinase Cz, since the process isindependent from Ca2+, resistant to PMA down modulation and since thekinase can be specifically immuno-recognized. In vivo, CK18 and CK8 arephosphorylated on serine and uPAR occupancy elicits a time-dependentincrease in phosphorylation of CK8, cell shape changes andredistribution of cytokeratin filaments. Pro-uPA treatment causedrounding up of the cells and reduction of their diameter. In controlcells, cytokeratins were distributed in a lattice array parallel to thesurface of the coverslips; upon treatment with pro-uPA, little or nocytokeratins were observed in filament form. These data tie together themigratory effect of pro-uPA, the activation of protein kinase C and thecell shape changes. Tyrosine-phosphorylation of a non-characterized 35kDa protein occurs upon binding of pro-uPA to U937 myeloid cells. Inthese cells, occupancy of uPAR promotes cell adhesion during PMA-induceddifferentiation. It has been shown that uPAR directly binds vitronectinand promotes cell adhesion. Moreover, uPAR directly interferes with thesubstrate recognition of adhesion receptors by forming complexesdirectly with the beta integrins, and hence promoting adhesion onvitronectin and inhibiting adhesion on fibronectin (Wei et al., 1996).Indeed, uPAR can be co-immunoprecipitated with anti-integrins IgG. Theeffect of uPAR on cell adhesion is also in line with its chemokine-likeactivity. A direct interaction of uPAR with integrins is suggested bytheir co-immunoprecipitation and by the finding of peptides thatspecifically inhibit the co-immunoprecipitation without interfering withthe binding of uPA or vitronectin to uPAR (Wei et al., 1996). Therefore,integrins might represent the described adaptor suggested by Resnati etal. (1996). In this respect, the uPAR-like small molecules to bedescribed in this application, have whatsoever no connection, eitherstructural or conceptual, with the peptide identified by Wei et al.(1996).

In monocytes and fibroblasts, occupancy of uPAR induces chemotaxis andthe effect does not require extracellular proteolysis (Resnati et al.,1996). In this system, ATF binding causes a time-dependent and transientactivation of a tyrosine kinase of the Src family, the p56/p58 Hck. Inaddition, uPAR itself associates with tyrosine kinases, through whatappears to be a transmembrane-adaptor-mediated mechanism. Indeed, cellslacking uPAR, and therefore not responsive to ATF, are capable ofchemotaxis when challenged with a mixture of chymotryptic fragments ofsoluble uPAR (chymotrypsin cleaves uPAR in two fragments between domainD1 and D2) (Resnati et al., 1996). This effect also occurs infibroblasts derived from uPAR−/−mice. Therefore, uPAR can recognize acell surface molecule which in turn is capable of mediating signaltransduction. The occupancy of surface uPAR must in fact expose bindingsites for the unidentified adaptor and hence transform uPAR from areceptor into a ligand. The role of tyrosine kinases in uPAR-dependentchemotaxis is also shown by the effect of tyrosine kinase inhibitorsthat prevent the chemotactic response to ATF, and by the failure ofprimary src−/−fibroblasts to respond to the soluble, cleaved uPAR(Resnati, Blasi and Fazioli, unpublished). Interestingly, while uPAbinding to uPAR is strictly species-specific, human uPAR acts as achemoattractant as efficiently on human as on murine cells. Inparticular, chymotrypsin-cleaved soluble uPAR has an IC50 of about 20 pM(Resnati et al., 1996).

The Chemoattractant Property of uPAR as a Target for Novel Drugs ActingDownstream of the uPA/uPAR Interaction

The uPA/uPAR system is involved in cancer cell invasiveness, and also ininflammation and other diseases (Danø et al., 1990). And in fact, theblock of uPA/uPAR interaction by specific antagonists, or the reductionof uPAR synthesis by antisense gene expression leads to a drasticreduction of the migratory properties of cancer cells (Fazioli andBlasi, 1994). In human cancer, the expression level of the components ofthe system, uPA, PAI-1 and uPAR, directly relates to the malignancy ofthe disease and its prognosis (Brünner et al., 1996). In fact, many ifnot all solid cancers have already released metastatic cells which arefound in the bone marrow. The presence of uPAR-positive metastatic cellsat the time of diagnosis represents a sign of extreme likelihood of poorprognosis, while the presence of metastatic cells per se does not.

On the basis of the above, uPAR appears to be a specific target fornovel types of drugs that might interfere with the process ofinflammation or of cancer invasiveness. Two different possibilities areopen: to block uPAR by interfering with uPA binding (uPAR antagonists),or to prevent the direct transduction of a migratory signal, viainteraction with other molecules, bypassing the step of the interactionbetween uPA and uPAR. Drugs preventing the binding of uPAR to the beforementioned adaptor should also prevent chemotaxis via uPAR. Therefore,antagonists of the uPAR/adaptor interaction should block or decrease themigration of cells and hence might be useful in diseases where thisdecrease or block is advantageous, like cancer, autoimmune andexcess-inflammatory reactions.

uPAR as Target for Drugs Enhancing the Recruitment of Inflammatory Cells

The recruitment of cells via the uPA-uPAR interaction suggests thatdrugs should be found which promote cell recruitment by binding to uPAR.And indeed, uPA derivatives like the amino terminal fragment ATF,pro-uPA or possibly also synthetic peptides that cover the uPAR-bindingregion of uPA are in fact capable of substituting for uPA in variousfunctions not requiring uPA proteolytic activity (see for example,Fazioli and Blasi, 1994; Besser et al., 1996, Resnati et al., 1996). Onthe other hand, since uPA binding to uPAR causes a conformational changethat transforms uPAR from a receptor for uPA into a ligand for a stillunidentified membrane adaptor (Resnati, et al. 1996), an alternativepossibility of enhancing cell recruitment would be to bypass the firststep, i.e. the uPA-uPAR binding step, and stimulate cell migration byacting at the level of the unidentified adaptor with uPAR itself orspecific uPAR-like agonists. These agonists might therefore be useful indiseases in which the recruitment of cells is blocked by genetic oracquired malfunctions upstream of the uPAR/adaptor interaction. Also,the availability of such drugs might be advantageous in connection withincreasing the immunogenicity of various antigens, for example invaccination, as it might act like an adjuvant by recruiting cellsresponsible of natural immunity.

uPAR as Target for Controlling HIV Infection

uPAR is an activation antigen in both T-lymphocytes and in monocytes(Nykjar et al., 1994; Bianchi et al., 1996), i.e. in cells capable ofactive migration and in which HIV can replicate. While CD3-positivecirculating lymphocytes do not or only weakly express uPAR, CD8+ Tlymphocytes of patients of AIDS and other viral diseases display a highexpression of uPAR, and in several of these patients as many as 80% oftheir T-lymphocytes are high expressers (Nykjar et al., 1994). Afraction of persons remain uninfected by HIV despite multiple high-risksexual exposure, and this is connected with the presence of HIVsuppressive facfors in their blood (Paxton et al., 1996). Severalchemokines have been identified as HIV-suppressive facfors produced byCD8+ T cells (Cocchi et al., 1996) and these chemokines represent noveltargets for AIDS therapy. Soluble forms of uPAR exist in the blood anduPAR is in fact cleaved in cell lines and in cancer tissues. Sincecleavage activates the chemotactic activity of soluble uPAR (Resnati etal., 1996), these soluble, cleaved forms might, like actual chemokines,also interfere with the infectivity or survival of HIV. If a cleavedform of soluble uPAR is also involved in HIV infectivity or resistance,it might represent a target for a novel type of therapy in cases ofAIDS.

uPAR as Target for Drugs Enhancing Wound Healing

Excessive fibrin deposits are observed in plasminogen-deficient,uPA-deficient and in the double uPAR/tPA-deficient mice and these miceare also deficient in skin wound healing (Carmeliet and Collen, 1996).Several mechanisms may be active in this phenomenon, includingchemotaxis, since uPAR is localized at the leading edge of migratingkeratinocytes during re-epithelization of mouse skin wounds. Theimportance of monocytes and macrophages in the process of wound healingmakes it even more likely that this process might be augmented by a uPARagonist capable of reproducing its chemoattractant property.

Novel Approach to Blocking uPAR

So far, approaches directed to block the effect of uPAR in cellmigration have been limited to the search of uPAR antagonists, i.e.drugs that prevent the binding of uPA to uPAR. In general, such drugsare also expected to inhibit cell surface proteolytic activity. However,binding of uPA to uPAR stimulates chemotaxis by transforming thismolecule from a receptor into a ligand for an adaptor (Resnati et al.,1996). Therefore, the identification of a small molecule that can mimicthe chemotactic activity of uPAR gives a new handle to control theuPAR-dependent reactions, independent of its occupancy by uPA. Inrelation to the present invention, such small molecules have beendiscovered and characterized. They act downstream of the uPA/uPARinteraction and therefore have no effect on cell surface proteolyticactivity. These small molecules can be applied both in cases ofdeficient cell recruitment and migration (as stimulators of theprocess), and in cases of excess cell recruitment and migration. In thelatter case, the uPAR-like small molecules can be employed to identifysuitable inhibitors of the uPAR-adaptor interaction.

uPA is essential for inflammatory cell recruitment, binds uPAR andinduces chemotaxis by direct signalling in a uPAR-dependent way. In thisfunction uPAR interacts with an unidentified adaptor molecule, and infact a mixture of chymotryptic fragments of soluble uPAR haschemoattracting activity. The present invention is based upon thediscovery of the mechanism through which uPAR activates chemotaxis.First the region of uPAR inducing chemotaxis was identified. As shown inexample 1, soluble fragments of uPAR were produced either by separationof chymotrypsin-cleaved uPAR, or by recombinant DNA technology, and wereused in a chemotaxis assay to locate the region of uPAR responsible forthe chemoattracting activity on THP-1 cells. Such a region (residues88-92 of uPAR) was localized in the linker region between domain D1 andD2. In fact, the chemotactic activity was found either in domain D1 orD2, whenever they included this linker region: carboxyterminally ofdomain D1, or aminoterminally of domain D2. In example 2, thechemotactic activity of uPAR was reproduced using synthetic peptidesincluding the 88-92 uPAR sequence of uPAR, on human THP-1 monocytoidcells (and murine LB6 fibroblastic cells). Synthetic molecules (peptides1 and 2) including the sequence SRSRY (ser-arg-ser-arg-tyr) (SEQ IDNO:7) had a very potent chemotactic activity at concentrations as low as0.1 pM. Control peptides had no activity, in particular a peptidecovering the carboxyterminal sequence deduced from uPAR cDNA, and apeptide having the same amino acid composition as peptide 1 but ascrambled sequence. In example 3, we show that peptide 1 stimulateschemotaxis through the same mechanism employed by the ligand uPA, or bythe mixture of chymotrypsin fragments of the soluble uPAR. Peptide 1, infact, activates Hck tyrosine kinase of THP-1 cells with the sametime-course and concentration dependence observed with the mixture ofchymotrypsin fragments of soluble uPAR. Example 4 shows how topractically identify inhibitors of uPAR by employing a simple panningassay using uPAR mimicking peptides, i.e. peptide 1 and 2 of theinvention.

One aspect of the present invention relates to a method of stimulatingor increasing the chemotactic activity of a cell, comprising adding achymotrypsin-cleaved suPAR peptide or a functional analogue of saidpeptide.

Within the scope of the uPAR peptides according to the present inventionare uPAR peptides derived from uPAR found in mammalian species, such ashuman, bovine, murine and rat.

In the present context, the term “functional analogue” means a peptidewith an amino acid sequence which is not identical to achymotrypsin-cleaved suPAR peptide, but which has a substantiallyidentical effect with respect to stimulating or increasing thechemotactic activity of a cell. This effect will typically be tested inan assay such as the one described in example 1. The increase inchemotactic activity in such an assay is preferably at least 100% ofcontrol, more preferably at least 200% of control, even more preferablyat least 400% of control.

Non-limiting examples of functional analogues of the human uPARchemotatactic activity are:

ser-arg-ser-arg-tyr (SEQ ID NO:7)

ser-arg-asn-arg-tyr (SEQ ID NO:8)

ser-arg-gly-arg-tyr (SEQ ID NO:9)

ser-gln-ser-arg-tyr (SEQ ID NO:10)

ser-gln-asn-arg-tyr (SEQ ID NO:11)

ser-gln-gly-arg-tyr (SEQ ID NO:12)

pro-arg-ser-arg-tyr (SEQ ID NO:13)

pro-arg-asn-arg-tyr (SEQ ID NO:14)

pro-arg-gly-arg-tyr (SEQ ID NO:15)

pro-gln-ser-arg-tyr (SEQ ID NO:16)

pro-gln-asn-arg-tyr (SEQ ID NO:17)

pro-gln-gly-arg-tyr (SEQ ID NO:18)

Other molecules which contain the above listed functional analogues andhave an effect in a chemotactic assay will also be functional analogueswithin the scope of the present invention, as well as peptides orpeptoids having a similar sequence (e.g. 400%-80% sequence identity,such as 60%, 66%, 75% or 80% sequence similarity with any of the listedpeptides) as any of the listed peptides and which exhibit an effect inthe assay.

Another aspect of the present invention thus relates to a peptoidanalogue of the peptide with the sequence SRSRY (SEQ ID NO:7) comprisingone or more non-naturally occurring amino acids. The peptoid analoguesaccording to the invention can for example be prepared by using peptidesynthesis methods, such as the method described by Merrifield. 1963. Thesynthesis may include any number of non-naturally occurring amino acidsand optionally also including one or more naturally occurring aminoacids in the preparation. The peptides and functional analogues of theinvention can be added, in an effective amount, directly to cells for toan individual, such as a human patient, who harbours the cells in whichchemotactic activity can be stimulated or increased.

A very important aspect of the present invention relates to peptides ofthe invention. Such peptides are a peptide with the sequence SRSRY (SEQID NO:7) or a functional analogue thereof and a peptide with thesequence AVTYSRSRYLEC (SEQ ID NO:1) and a peptide with the sequenceSRSRYLEC (SEQ ID NO:3).

A particularly preferred embodiment of the present invention relates toa recombinant fusion protein, comprising one or more of the peptidespecies according to the invention and optionally additionallycomprising one or more of the peptide species according to theinvention.

Functional analogues, according to the invention, which are preparedusing peptide synthesis are within the scope of the present invention.Functional analogues, according to the invention, which are prepared byusing other methods than peptide synthesis, such as recombinantexpression, are also within the scope of the present invention.

A further aspect of the present invention is a method of stimulating orincreasing a local inflammatory response, comprising adding achymotrypsin-cleaved fragment, or mixture of fragments, of uPAR, saidfragment or mixture of fragments comprising an SRSRY (SEQ ID NO:7)peptide, or a functional analogue of said peptide.

In the present context, the term “stimulating or increasing localinflammatory response” means that one or more characteristics of aninflammatory response can be measured to be stimulated or increased.Such characteristics will typically be release of cytokines byT-lymphocytes or monocyte-macrophages, increase of chemotactic activity,ability to cause a local recruitment of lymphocytes/monocytes uponsubcutaneous administration in a suitable laboratory animal, etc. Inthis particular case, the use of uPA-deficient mice is particularlyfavourable as these mice have a very low anti-inflammatory response.Methods to measure these activities are available routinely, andincreases of such activities as mentioned of at least 50% are consideredsignificant, preferably at least 80% even more preferably at least 100%.

Another important aspect of the present invention is a method ofstimulating or increasing the chemotactic activity of a cell, comprisingadding a peptide comprising the sequence SRSRY (SEQ ID NO:7)or afunctional analogue of said peptide.

A further aspect of the present invention is a method of stimulating orincreasing wound healing, comprising adding a chymotrypsin-cleaved suPARpeptide or a functional analogue of said peptide. A peptide comprisingthe sequence SRSRY (SEQ ID NO:7) or a functional analogue of saidpeptide can be added according to the invention.

In the present context, the term “stimulating or increasing woundhealing” means that at least one of the physiological characteristics orprocesses involved in healing of various types of wounds in anindividual, such as a human, is substantially stimulated or increased.One way of quantifying such stimulation or increase in wound healingcomprises measuring wound healing in a plasminogen-deficient or even anormal mouse, and identifying the time required for 50% healing. Areduction of the time of healing, upon addition of an effective amountof a composition comprising suPAR peptide, of at least 20%, preferablyat least 50%, would represent a substantial wound healing stimulation.

An important aspect of the present invention relates to a method ofstimulating the kinase activity of p56/p59^(hck) comprising adding achymotrypsin-cleaved suPAR peptide or a functional analogue of saidpeptide or comprising adding a peptide comprising the sequence SRSRY(SEQ ID NO:7) or a functional analogue of said peptide.

In the present context, the term “stimulating the kinase activity” meanssubstantially increasing the ability of p56/p59^(hck) to phosphorylate aprotein substrate. This will typically be measured using in vitro assaysas the ones described in example 3. Quantification of such an effectmight be obtained by cutting the bands from a gel such as the one showedin FIG. 5, and subsequently measuring the 32P-radioactivity, afternormalization.

A very important aspect of the present invention relates to a method oftesting whether a compound is capable of stimulating the binding betweena chymotrypsin-cleaved soluble form of u-PAR and a cellular adaptor,said method comprising adding an effective amount of the compound to atest system comprising cells lacking endogenous u-PAR and measuringchemotactic activity; if chemotactic activity is present, then thecompound is capable of stimulating said binding.

In the present context the term “stimulating the binding” should betaken to mean increasing the binding in an assay, substantiallyresembling the above described, by at least 20%, preferably at least30%, more preferably by at least 40%, even more preferably by at least60%.

In another aspect, the present invention relates to a compound which iscapable of stimulating the binding between a chymotrypsin-cleavedsoluble form of uPAR and a cellular adaptor which has been selected bythe method of the invention as described above.

A particularly important aspect of the present invention relates to amethod of testing whether a compound is capable of blocking orinhibiting the binding between a chymotrypsin-cleaved soluble form ofuPAR and a cellular adaptor, said method comprising addingchymotrypsin-cleaved soluble form of uPAR and an effective amount of thecompound to a test system comprising cells lacking endogenous uPAR andmeasuring chemotactic activity. If chemotactic activity is inhibited orprevented, then the compound is capable of blocking said binding.

In the present context the term “blocking or inhibiting the binding”should be taken to mean decreasing the binding in an assay,substantially resembling the above described, by at least 20%,preferably at least 30%, more preferably by at least 40%, even morepreferably by at least 60%.

In a further aspect, the present invention relates to a compound whichis capable of blocking or inhibiting the binding between achymotrypsin-cleaved soluble form of uPAR and a cellular adaptor whichhas been selected by the method of the invention as described above.

An important embodiment of the present invention is a compound accordingto the invention for the treatment of cancer, autoimmune disease and/orhyper-inflammatory diseases. A compound which is capable of blocking orinhibiting the binding between a chymotrypsin-cleaved soluble form ofuPAR and a cellular adaptor would according to the invention beadministered to an individual, such as a human patient, in need thereofin a pharmaceutically acceptable form and in a pharmaceuticallyeffective amount.

A very important aspect of the present invention relates to a method ofscreening compounds for potential anti-inflammatory or anti-migratoryproperties. The method according to the invention comprises

(a) coating a plastic surface which does not normally bind mammaliancells with an effective amount of a peptide according to the invention,

(b) exposing selected mammalian cells to the coated surface for 30-300minutes at 37 C., in the presence or absence of a potentially inhibitorycompound, and

(c) testing the ability of the cells to bind to the peptide coatedsurface by microscopic inspection or Coomassie Blue staining of theplates; if cellular binding is inhibited or prevented, then the compoundis capable of blocking said binding and is a potential anti-inflammatoryor anti-migratory agent.

In a further aspect, the present invention relates to oligo nucleotides,in particular oligonucleotides encoding the peptides given by SEQ ID NO:1 and SEQ ID NO: 3.

Another aspect of the present invention relates to the use of anoligonucleotide according to the invention, for the recombinantexpression of peptides for use in the screening of compounds accordingto the invention.

Preferred embodiments of the present invention relate to use ofoligonucleotides according to the invention, wherein the recombinantexpression is performed in an expression system chosen from the groupconsisting of an E. coli expression system, a yeast expression system, amammalian expression system or an insect cell expression system.

When using the oligonucleotides according to the invention as the basisfor preparing peptides according to the invention by performingrecombinant expression, it is preferred to combine said oligonucleotides with nucleic acid sequences, such as regulatory sequences,which enable. the expression system of choice to express large amountsof peptide.

A particularly important aspect of the present invention relates to amethod of stimulating or increasing anti-tumour immunity in autologousbone marrow transplantation treatment of an individual, such as a humanpatient, bearing tumours, comprising modifying tumour cells bytransfecting them with a eukaryotic vector expressing a nucleic acidsequence encoding the peptide SRSRY (SEQ ID NO:7), or a functionalanalogue thereof.

In the present context, the term “anti-tumor immunity” means the set ofcellular and chemical reactions which an organism can organize with theaim of rejecting a tumor. In this respect, the production of cytokinesand chemokines is of major importance as it will drive the recruitmentand the functional activation of cells that can specifically attack thetumor cells and hence destroy it.

Another important aspect of the present invention relates to a method ofstimulating or increasing cellular immunity in an individual, such as ahuman patient, who is immunodeficient, said method comprising adding achymotrypsin-cleaved suPAR, said suPAR comprising the peptide SRSRY (SEQID NO:7), or a functional analogue thereof.

In the present context, the term “cellular immunity” means therecruitment and the functional activation of immune cells, such asT-lymphocytes, NK-cells, B-lymphocytes, macrophages etc.

In the present context, the term “immunodeficient” means that the immunesystem of an individual is compromised and thus can be said to functionat a sub-normal level with respect to at least some of the systemsreactions.

Yet another aspect of the present invention relates to a method ofvaccinating a subject, such as a human patient, suffering from tumours,with the aim of stimulating an anti-tumour T cell response, said methodcomprising vaccination of said subject with a human tumour cell linecompatible on both the histotype and the HLA-A2 basis and modified so asto release the chemotactic region of the human or murine or bovine orrat uPAR, said chemotactic region being a fusion peptide or a domain ofuPAR containing the SRSRY (SEQ ID NO:7) peptide or a functional analoguethereof.

A preferred embodiment of the present invention relates to a methodaccording to invention, wherein vaccination is carried out by usingautologous tumor cells.

A very important aspect of the present invention relates to a method ofexploiting the cellular immunity of uPAR derivatives or peptides of thepresent invention to kill tumour cells, said method comprising injectinga tumour-bearing subject, such as a human patient, with tumour-targetedcells modified to express the chemotactic region of uPAR from human,bovine, murine or rat, or any other uPAR having substantially the sameproperties, said method further comprising fusing the target vector tosaid region, being a peptide or a domain of uPAR containing the SRSRY(SEQ ID NO:7) peptide, or a functional analogue thereof.

In the present context the term “uPAR derivatives” is used as defined inWO 90/12091, whereas the term “tumour-targeted cells” means cells that,naturally or because they are specially engineered, can be injected intoan individual and will specifically interact with tumour cells of saidindividual.

LEGENDS TO FIGURES

FIG. 1 Part A of this figure shows the sequence of active peptides 1(SEQ ID NO:1) and 2 (SEQ ID NO:2) reproducing sequences of human uPAR,and the scrambled version of peptide 1 (SEQ ID NO:4) that was employedas a negative control employed in Example 2. Obviously the sequenceSRSRYLEC (SEQ ID NO:3) is in common between the peptides 1 and 2.However, the active region can be further delimited since experimentsfrom Example 1 show that the presence of the sequence SRSRY (SEQ IDNO:7) either carboxyterminal to domain D1, or aminoterminal to domainD2,3 confers to these peptides an otherwise absent chemotactic activity.The sequence SRSRY (SEQ ID NO:7) is present in human uPAR betweenpositions 88 and 92. Part B of this figure shows the sequence of theactive peptide region in rat (SEQ ID NO:15), mouse (SEQ ID NO:7) andbovine (SEQ ID NO:8) uPAR and its comparison with that of human uPAR. Italso shows a preliminary consensus sequence that indicates that thefourth and fifth residues are always conserved, the first and secondresidues show a conservative change while the third residue appears tobe more freely substituted.

FIG. 2. This figure shows a scheme depicting the structure of varioussoluble forms of uPAR to be employed in Example 1. Schematicrepresentation of the soluble three-domain uPAR (D123) and the fourdeletion mutants. The number indicate amino acids residues according tothe human uPAR sequence deduced from cDNA (Roldan et al., 1990) using asnumber one the first residue after the signal sequence. Each constructwas named on the basis of which of the three domains was still retained:D123 is the symbol for suPAR containing residues 1-274 and beingsecreted in a soluble form. Each construct contains at the 3′ end aFLAGTM (SEQ ID NO:6) epitope which is employed for purification of theproteins onto a commercial antibody-affinity column.

FIG. 3. This figure shows the chemotactic response of THP-1 cells tofragments or mutants of soluble uPAR. For each set of experiments,migration toward the assay medium served as control (random cellmigration) and is referred to as 100% migration. All experiments wereperformed in triplicate; the number of cells counted per high powerfield (ten fields for each condition) is expressed as percent of thecontrol values. The proteins were purified as described in the Methodssection. Data points represent the mean of three independent experiments(+SEM). The constructs used are depicted in FIG. 2.

FIG. 4. In this figure, the chemotactic effect of synthetic peptides onTHP-1 cells is shown. Peptides 1 and 2 show very potent activity, whilepeptide 3 or peptide-1 scrambled do not. Dose-dependent chemotacticresponse of THP-1 cells to peptide 1 (AVTYSRSRYLEC (SEQ ID NO:1), (aa84-95) (panel A), peptide YTARLWGGTLLT (SEQ ID NO:2) (aa 302-313 ofuPAR) (panel B), peptide (SRSRYLEC (SEQ ID NO:3), aa 88-95 of uPAR)(panel C) corresponding to the uPAR sequences of Roldan et al. (1990);panel D shows the chemotactic response to peptide TLVEYYSRASCR (SEQ IDNO:4), a scrambled version of peptide 1.

FIG. 5. In this figure, the stimulating effect of peptide 1 on theactivity of Hck kinase of THP-1 cells is shown. THP-1 cells weremetabolically labelled with [35S]-TransLabel, acid washed, and eithermock-treated or treated with 0.1 nM peptide 1 (AVTYSRSRYLEC, (SEQ IDNO:1)) for the indicated time at 37° C. Radiolabelled cell lysates wereimmunoprecipitated (Resnati et al., 1996) using an affinity-purifiedrabbit polyclonal anti-p56/p59hck antibody.

(A) An aliquot from each immunoprecipitate was directly analyzed bySDS-PAGE to control that equal amount of p56/p59hck were present duringin vitro kinase assay.

(B) The remainder of each immunoprecipitate was subjected to in vitrokinase assay in presence of 5-10 mCi of [g-32P]ATP (˜3000 Ci/mmol,Amersham) and 5 mg rabbit muscle enolase for 5 minutes at roomtemperature. Eluates were thus analyzed by SDS-PAGE autobiography (7.5%acrylamide).

EXAMPLE 1

Chemotactic Response of Human or Murine Cells Stimulated with Fragmentsof Soluble uPAR or of Soluble Recombinant uPAR Mutants

Methods

Cloning of Soluble uPAR Mutants

In the following description, aminoacid residues are numbered accordingto the previously published uPAR CDNA sequence (Roldan et al., 1990).Mutant cDNA's encoding soluble uPA receptors were generated byPolymerase chain reaction (PCR). The following oligonucleotides wereused for the various constructs:

Construct D1 (1-92): oligonucleotides FRA18 and D1.3′T.

Construct D12 (1-191): oligonucleotides FRA18 and D2.3′T

Construct D123 (1-274): oligonucleotides FRA18 and D3.3′T.

The PCR-products were digested with BclI and ClaI, and cloned inBluescript SK− (Stratagene) digested with BamHI and ClaI.

To generate the constructs encoding D2 (93-191) and D23 (93-274) the D1region in the D12 and D123 constructs was deleted by substituting theNruI/NsiI fragment containing the D1 coding region, with a fragmentgenerated by amplifying the uPAR cDNA with the primers FRAl18 and D2.5′Tand digested with the same enzymes. To generate the construct encodingD3 (192-275), the D12 region in the D123 construct was deleted bysubstituting the NruI/NcoI fragment containing the D12 coding region,with a fragment generated by amplifying the uPAR cDNA with the primersFRA18 and D3.5′T and digesting with the same enzymes.

All the mutant receptors were tagged at the carboxyterminus with thepeptide sequence HRRASVDYKDDDDK (SEQ ID NO:5) which includes the proteinkinase substrate and the FLAGTM epitope by inserting in the carboxyterminal Clal site a linker made by annealing the two oligonucleotidesK/FOcs and K/FOas. This linker was inserted into the ClaI site locatedat the carboxyterminus of all the constructs. All the recombinant codingregions were amplified with the primers NS1 and 46D, digested with NcoIand transferred to the eukaryotic expression vector pBNSEN digested withNcoI and EcoRV. Oligonucleotides (5′-3′):

FRA18: ATTATACTCGAGGAAGACGTGCAGGGACCCCGCGCA; (SEQ ID NO:19)

D1.3′T: TTATCGATGGTAACGGCTTCGGGAATA; (SEQ ID NO:20)

D2.3′T: TTATCGATGGCCATTCTGCGGCAGATT; (SEQ ID NO:21)

D3.3′T: TTATCGATGTGGGTGGTTACAGCCACT; (SEQ ID NO:22)

D2.5′T: AATGCATTCGAGGCCCCAAGAGGCTGGGA; (SEQ ID NO:23)

D3.5′T: TCCATGGGTGCTGTTCCCCTTGCAGCTGTAACACTGGCGGCCCCAAGAGGCTGGGA; (SEQID NO:24)

K/FOcs: CGACGAGCATCTGTCGACTATAAGGATGACGACGACAAGTAA; (SEQ ID NO:25)

K/FOas: CGTTACTTGTCGTCGTCATCCTTATAGTCGACAGATGCTCGT; (SEQ ID NO:26)

NS1: CCGCGGAAGAACCCATGGGACTCCCAA; (SEQ ID NO:27)

46D: CAAGCTTACTTGTCGTCGTCATCC. (SEQ ID NO:28)

Expression and purification of recombinant proteins Semi-confluent COS7cells were harvested in PBS containing 1 mM EDTA and washed twice withRPMI medium. 0.8 ml cell suspension (1-2×107 cells/ml in RPMI) wastransferred to electroporation cuvettes (0.4 cm, Bio-Rad) containing theplasmid DNA (30 μg, 1 mg/ml in water) and electroporated at 960 μF, 240V in an electroporafor (GenePulser, Bio-Rad). After electroporation,cells were transferred to a T175 flask containing 30 ml of rich medium(DMEM with 100% FCS). The next morning, cells were washed three timeswith PBS and supplemented with 50 ml of serum free medium (DMEMcontaining 1 % Nutridoma NS, Boehringer Mannheim). Every 4-5 days,conditioned medium was collected and fresh medium added. Recombinantproteins were purified from the conditioned medium by passage over ananti-FLAGTM affinity column (M2 Affinity gel, Sigma). After washing withPBS, recombinant proteins were eluted with 0.1 M glycine pH 3.0.

Purified D1 (1-87) and D23 (88-274) were prepared by cleavage of D123with chymotrypsin (Behrendt et al. 1991) followed by passage over theFLAGTM affinity column. D1(1-87) was recovered in the flow-through whilethe D23(88-274) containing the FLAGTM epitope was retained and latereluted with 0.1 M glycine pH 3.0.

Chemotaxis Assay

Chemotaxis analysis were performed using modified Boyden chamberscontaining polyvinylpyrrolidone polycarbonate filters (13 mm diameter, 5mm pore size) coated with collagen type I (100 mg/ml in PBS pH 7.4). Thecoated filters were washed with medium supplemented with 0.2% bovineserum albumin (BSA) and then placed in the Boyden apparatus. After acidwash, 2×105 THP-1 cells suspended in serum-free medium were added abovethe filter in the Boyden chamber. Purified mutants were diluted inserum-free medium at the indicated concentrations and added below thefilter in the lower chamber. The chambers were incubated in a humidifiedincubafor at 37° C. in 5% CO2 in air for 90 minutes. Filters wereremoved, the upper surface scraped free of cells, fixed in methanol andstained with crystal violet. For each set of experiments, migrationtoward the assay medium served as control (random cell migration) and isreferred to as 100% migration. All experiments were performed intriplicate; data are reported as number of cells counted for high powerfield (ten fields for each condition) and expressed as percentage of thecontrol values. Data points represent the mean of three independentexperiments (+SEM). When cells of the fibroblast-type or cancer cellswere employed for the assay, the time required for migration was higherand the Boyden chamber were usually incubated overnight.

Results

FIG. 2 shows a schematic representation of the structure of recombinantsoluble uPAR and of various mutants generated. Previous studies haveshown that a soluble form of uPAR (suPAR) is a potent chemoattractant indifferent cell lines, also in cells lacking endogenous uPAR (Resnati, etal., 1996). However, activity of suPAR requires chymotrypsin cleavage atTyr87, between the N-terminal, ligand binding, domain D1 and domainD2+D3. This suggests that a unique conformation, uncovered bychymotrypsin cleavage, is required and must interact with a yetunidentified cell-surface receptor which might act as a transmembraneadaptor. The same mechanism applies to uPA binding to thesurface-anchored uPAR.

In order to identify the region of uPAR endowed with chemoattractantactivity, we first defined the domain of uPAR responsible for thesignalling effect and thus for the interaction with the transmembraneadaptor. We engineered and purified different soluble uPAR mutants andanalyzed them for the ability to stimulate chemotaxis on THP-1 cells. Itis known that truncation of the carboxy-terminal domain results in theloss of the membrane-anchoring capacity and thus in the secretion of anotherwise active molecule (Masucci et al., 1991); to produce solubleforms of uPAR, the sequence corresponding to aa 275-313 (Roldan et al.,1990) was omitted in all the constructs. FIG. 2 shows the schematicrepresentation of the three-domains entire suPAR (D123) (aa 1-274) andof the four deletion mutants constructed: D23 (aa 93-274), D1 (aa1-92),D2 (aa 93-191) and D3 (aa192-274). A kinase-site, followed by anin-frame FLAGTM epitope was inserted at the C terminus of each mutant.All the constructs were cloned in a modified form of the pBNSENexpression vector, COS7 cells transfected by the calcium phosphateco-precipitation method and secreted uPAR mutants protein purified byaffinity chromatography. As a control, the purified D123 protein wasdigested with chymotrypsin (Resnati et al., 1996) and the correspondingproducts, D1 (aa 1-87) and D23 (88-274), were separated by an additionalround of purification on FLAGTM antibody columns. The details ofconstructs preparation, the purification method and the system employedto assay chemotaxis are given in the above methods section.

FIG. 3 shows the results of the chemotaxis assays employing derivativesof suPAR D123 as chemoattractants. As predicted by previous results(Resnati et al., 1996), purified uncleaved DI23 was unable to stimulatechemotaxis in THP-1 cells, while chymotrypsin cleavage of this proteinconferred a strong chemoattractant property (data not shown). When theproducts of chymotrypsin cleavage of D123 were purified and analyzedseparately, only the D23 fragment (FIG. 3, panel F) (residues 88-274)was able to elicit a dose-dependent chemotactic response, while D1(1-87) (FIG. 3, panel E) did not influence THP-1 cell migration. Howeverwhen the domain D1 was produced by recombinant DNA technology (D1-92)was tested, it was unexpectedly found to induce a dose-dependentchemotactic effect (FIG. 3, panel A). The sequence of soluble D1comprised residues 88-92, while the chymotryptic fragment comprised onlythe sequence 1-87. Likewise, the recombinant D23 (93-274) failed toinduce chemotaxis through the entire concentration range analyzed (FIG.3, panel B). It was noticed that the sequence 88-92 was missing in thismutant, while it was present in the chymotryptic D23(88-274) fragment.These findings overall indicate that the sequence from amino acid 88 to92, SRSRY (SEQ ID NO:7), was responsible for the chemotactic effect onTHP-1 cell migration. Accordingly, no effect on cell migration wasobserved when the assay was performed in the presence of comparabledoses of purified domain D2(93-191) (FIG. 3, panel C) and D3(192-274)(FIG. 3, panel D), in which the sequence SRSRY was not present. Thisresult was obtained not only with THP-1 cells, but also with other cellslike murine 3T3 fibroblasts and murine LB6 cells (data not shown).Therefore the chemotactic effect of uPAR fragments is notspecies-specific, unlike the binding specificity of uPA to uPAR.

EXAMPLE 2

Chemotactic Response of Human Cells Stimulated with Peptides Derivedfrom Human uPAR

To confirm the hypothesis that the sequence SRSRY (SEQ ID NO:7) of humanuPAR was responsible for the uPAR-mediated signalling effect and thusfor the interaction with one or more adaptors, another set ofexperiments was performed. Three human uPAR-related peptides wereanalyzed as inducers of chemotaxis on THP-1 cells: peptide 1(AVTYSRSRYLEC (SEQ ID NO:1); amino acid sequence 84-95 of human uPAR)and its shorter analogue peptide 2 (SRSRYLEC (SEQ ID NO:3), amino acidsequence 88-95 of human uPAR) were tested as inducers of chemotaxis onTHP-1 cells. Numbering of sequence positions follows that of Roldan etal. (1990), in which amino acid residue 1 is the first residue after thesignal peptide. The two above peptides both contain the SRSRY (SEQ IDNO:7) motif. The third peptide was chosen in the carboxy terminal regionof the uPAR cDNA sequence (YTARLWGGTLLT (SEQ ID NO:2), aa 301-313) andused in the assay as a negative control. An additional peptide wasemployed to control for the specificity of the effects mediated by theSRSRY (SEQ ID NO:7)-containing peptides. More precisely, a scrambledversion of peptide 1 (TLVEYYSPASCR (SEQ ID NO:4)) was also tested underthe same experimental conditions. Results of these experiments are shownin FIG. 4. Peptides 1 and 2 were able to elicit a dose-dependentchemotactic effect, with a maximal response at concentrations as low as0.1 pM (FIG. 4, Panels A and C). On the contrary, when the assay wasperformed in the presence of similar doses of the control peptide 3, orof peptide TLVEYYSRASCR (SEQ ID NO:4) (peptide 1 in a scrambledsequence), no effect on cell migration was observed over the entireconcentration range analyzed (FIG. 4, panels B and D). Thus, peptidescontaining the motif SRSRY (SEQ ID NO:7) of human uPAR have a potentchemotactic activity and must be responsible for uPAR induced migration.

These peptides represent novel chemotactic agents acting downstream ofthe uPA/uPAR interaction.

EXAMPLE 3

Peptide 1 (AVTYSRSRYLEC (SEQ ID NO:1)) Affects Chemotaxis Activatingp56/p59hck Tyrosine Knase Activity in THP-1 Cells

The mixture of chymotrypsin fragments of suPAR, like the uPA/uPARinteraction, induces chemotaxis by transiently activating the p56/p59hckprotein kinase (Resnati et al., 1996). If human uPAR-related peptidescontaining the SRSRY (SEQ ID NO:7) motif induce a chemotactic responseon THP-1 cells though the same mechanism exploited by the uPA/uPARinteraction (i.e. by binding to the unidentified adaptor), they shouldalso activate p56/p59hck. THP-1 cells were metabolically labelled with[35S]-TransLabel, acid washed, and either mock-treated or treated with0.1 nM peptide 1 (AVTYSRSRYLEC (SEQ ID NO:1)) for the indicated time at37° C. Radiolabelled cell lysates were immunoprecipitated (Resnati etal.,1996) using an affinity-purified rabbit polyclonal anti-p56/p59hckantibody. An aliquot from each immunoprecipitate was directly analyzedby SDS-PAGE to control that equal amount of p56/p59hck were presentduring in vitro kinase assay (FIG. 5, panel A). The remainder of eachimmunoprecipitate was subjected to in vitro kinase assay in presence of5-10 mCi of [g-32P)]ATP (˜3000 Ci/mmol, Amersham) and 5 mg rabbit muscleenolase (substrate) for 5 minutes at room temperature. Eluates were thusanalyzed by SDS-PAGE autobiography (7.5% acrylamide). As shown in FIG.5, panel B, an increase in autophosphorylation of p56/p59hck, as well asan increase of phosphorylation of the exogenous substrate, was observedafter peptide 1 stimulation. The effect was very rapid, since p56/pS9hckstimulation was already- evident after 2 minutes, reached a maximumafter minutes and returned to the basal level 30 minutes after theaddition of the peptide. No effect on p56/p59hck activity was observedwhen 0.1 nM of the corresponding scrambled peptide was used in the assay(data not shown). The time-course of the effect is identical to thatobserved with the addition of ATF or of chymotryptic uPAR fragments toTHP-1 cells and reported previously (Resnati et al., 1996).

EXAMPLE 4

Panning with uPAR-mimicking Peptide and Selection of Inhibitors Thereof

Mammalian cells adhere on specially coated plastic plates, but do notadhere when plated onto plastic dishes commonly employed to growbacteria. However, when those plates are coated with peptide 1 or 2 (ordomain D1-1-92) or with recombinant forms of these peptides, they adherebecause they express on their surface specific D1(1-92)-bindingproteins. The adhesion can be measured.

As a positive control (i.e. to set up the technique), cells (106)exposing a high number of uPAR molecules are used and the panning iscarried out on plates coated with a specific anti-uPAR antibody. As anegative control, the same cells but now not expressing uPAR areemployed. The cells employed as negative controls are LB6, 32D, 3T3fibroblasts, B-lymphoid cell lines and others. As positive controls,THP-1 cells, LB6/uPAR, 32D/uPAR and other expressing cell lines areemployed.

For the screening assay, the same cells employed as negative controls(106) are used, and the plates are coated with domain D1(1-92) or domainD1(1-87) (control). The ability of the cells to bind to the plates istested after 30-300 minutes incubation at 37° C. by microscopicinspection, or by staining the plates with Coomassie Blue. The same canbe done using different techniques.

The ability of peptides 1- or 2-coated plates to specifically bind theTHP-1 cells is the basis for the screening technique, as any compoundcapable of inhibiting this binding is a potential antiinflammatoryagent, inhibiting chemotaxis. For example, peptide 1 or peptide 2 can infact inhibit adhesion of cells onto D1(1-92), while the scrambled orother peptides cannot. Such compounds, therefore, can be tested asinhibitors of chemotaxis, employing the methods outlined in examples1-3. Coating can also be carried out with other recombinant forms of theSRSRY (SEQ ID NO:7) sequence in which the epitope is synthesized infusion with other proteins. Suitable proteins are ferritin (Sidoli etal. (1993)), thioredoxin, GST, beta galactosidase, human serum albumin.

Inhibitors can be selected from phage display libraries of peptides,branched tripeptides combinatorial libraries, libraries of natural or ofchemical compounds, and any other potentially exploitable collection ofcompounds.

REFERENCES

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5 Brünner, N., Holst-Hansen, C., Pedersen, A. N., Pyke, C.,Høyer-Hansen, G., Foekens, J. and Danø, K. (1996). Urokinase plasminogenactivafor receptor in breast cancer. In “Breast Cancer: Adv. in Biol.and Therap.”,

6 F. Calvo, M. Crepin and H. Magdelenat, Eds. John Libbey Eurotext, p.201-207.

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14 Gyetko, M. R., Chen, G.-H., McDonald, R. A., Goodman, R., Huffnagle,G. B., Wilkinson, C. C., Fuller, J. A. and Toews, G. B. 1996. Urokinaseis required for the pulmonary inflammatory response to Cryptococcusneoformans. J. Clin. Invest., 97, 1818-1826.

15 Gyetko, M. R., Todd III, R. F., Wilkinson, C. C. and Sitrin, R. G.1994. The urokinase receptor is required for human monocyte chemotaxisin vitro. J. Clin. Invest., 93, 1380-1387.

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22 Roldan, A. L., Cubellis, M. V., Masucci, M. T., Behrendt, N., Lund,L. R., Danø, K., Appella, E., and Blasi, F. 1990. Cloning and expressionof the receptor for human urokinase plasminogen activator, a centralmolecule in cell-surface plasmin-directed p-oteolysis. EMBO J. 9,467-470.

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28 1 12 PRT Artificial Sequence Description of ArtificialSequencesynthetic peptide analogue of the human uPAR 1 Ala Val Thr TyrSer Arg Ser Arg Tyr Leu Glu Cys 1 5 10 2 12 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 2 Tyr Thr Ala Arg Leu Trp Gly Gly Thr Leu Leu Thr 1 5 10 3 8PRT Artificial Sequence Description of Artificial Sequencesyntheticpeptide analogue of the human uPAR 3 Ser Arg Ser Arg Tyr Leu Glu Cys 1 54 12 PRT Artificial Sequence Description of Artificial Sequencesyntheticpeptide analogue of the human uPAR 4 Thr Leu Val Glu Tyr Tyr Ser Arg AlaSer Cys Arg 1 5 10 5 14 PRT Artificial Sequence Description ofArtificial Sequencesynthetic peptide analogue of the human uPAR 5 HisArg Arg Ala Ser Val Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 10 6 6 PRTArtificial Sequence Description of Artificial Sequencesynthetic peptideanalogue of the human uPAR 6 Phe Leu Ala Gly Thr Met 1 5 7 5 PRTArtificial Sequence Description of Artificial Sequencesynthetic peptideanalogue of the human uPAR 7 Ser Arg Ser Arg Tyr 1 5 8 5 PRT ArtificialSequence Description of Artificial Sequencesynthetic peptide analogue ofthe human uPAR 8 Ser Arg Asn Arg Tyr 1 5 9 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 9 Ser Arg Gly Arg Tyr 1 5 10 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 10 Ser Gln Ser Arg Tyr 1 5 11 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 11 Ser Gln Asn Arg Tyr 1 5 12 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 12 Ser Gln Gly Arg Tyr 1 5 13 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 13 Pro Arg Ser Arg Tyr 1 5 14 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 14 Pro Arg Asn Arg Tyr 1 5 15 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 15 Pro Arg Gly Arg Tyr 1 5 16 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 16 Pro Gln Ser Arg Tyr 1 5 17 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 17 Pro Gln Asn Arg Tyr 1 5 18 5 PRT Artificial SequenceDescription of Artificial Sequencesynthetic peptide analogue of thehuman uPAR 18 Pro Gln Gly Arg Tyr 1 5 19 36 DNA Artificial SequenceDescription of Artificial Sequencesynthetic DNA 19 attatactcg aggaagacgtgcagggaccc cgcgca 36 20 27 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 20 ttatcgatgg taacggcttc gggaata 27 2127 DNA Artificial Sequence Description of Artificial SequencesyntheticDNA 21 ttatcgatgg ccattctgcg gcagatt 27 22 27 DNA Artificial SequenceDescription of Artificial Sequencesynthetic DNA 22 ttatcgatgt gggtggttacagccact 27 23 29 DNA Artificial Sequence Description of ArtificialSequencesynthetic DNA 23 aatgcattcg aggccccaag aggctggga 29 24 56 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 24tccatgggtg ctgttcccct tgcagctgta acactggcgg ccccaagagg ctggga 56 25 42DNA Artificial Sequence Description of Artificial Sequencesynthetic DNA25 cgacgagcat ctgtcgacta taaggatgac gacgacaagt aa 42 26 42 DNAArtificial Sequence Description of Artificial Sequencesynthetic DNA 26cgttacttgt cgtcgtcatc cttatagtcg acagatgctc gt 42 27 27 DNA ArtificialSequence Description of Artificial Sequencesynthetic DNA 27 ccgcggaagaacccatggga ctcccaa 27 28 24 DNA Artificial Sequence Description ofArtificial Sequencesynthetic DNA 28 caagcttact tgtcgtcgtc atcc 24

What is claimed is:
 1. An isolated peptide consisting of the sequenceAVTYSRSRYLEC (SEQ ID NO: 1).
 2. An isolated peptide consisting of thesequence SRSRYLEC (SEQ ID NO: 3).
 3. An isolated peptide consisting ofthe sequence SRSRY (SEQ ID NO: 7).
 4. A recombinant fusion protein,comprising the peptide of claim
 1. 5. A recombinant fusion protein,consisting of a peptide consisting of the sequence of SEQ ID NO: 1 and apeptide consisting of the sequence of SEQ ID NO:3.
 6. A method ofincreasing the chemotactic activity of a cell, comprising adding to theenvironment of said cell a peptide consisting of the sequence SRSRY (SEQID NO: 7) at a dosage of 1.0 nM or less, whereby the chemotacticactivity of said cell is increased at least 100% with respect to thechemotactic activity of said cell in the absence of said peptide.